Project Summary The proposed research will interrogate natural product scaffolds as starting points for antivirulence and narrow-spectrum agents. Of specific interest are compounds that mitigate bacterial biofilms, which are the causative agent in hospital-acquired infections, the development of resistance in bacteria, the rejection of medical implants, and many other health related diseases. The compounds, which this proposal will focus on, have been chosen from privileged areas where bacteria utilize chemical warfare to prevent colonization of invading species. Here we present a multi-faceted approach, including organic synthesis, molecular genetics, proteomics, transcriptomics, and microbiological assays that begins with these privileged scaffolds but has as an overarching goal of developing next generation therapeutics and tool compounds to better understand processes within a multispecies environment. The first two research thrusts seek to identify antivirulence compounds that inhibit one of the following processes: biofilm formation, quorum sensing, production of virulence factors, etc. Starting with known antifoulant and signaling molecules, we will explore the unique chemical features that endow each molecule with their biological effect. Furthermore, we will utilize the chemical toolbox to design compounds with improved chemical and physical properties to improve the likelihood of translation. Analogs will then be tested with the goal of identifying specific processes that the natural product affects and allow for a broader evaluation of the target in general biofilm processes. The third research thrust involves the chemical synthesis of natural product scaffolds identified in the rhizosphere. We have recently shown that these compounds possess species-specific activity, which is ideal for the development of ?narrow-spectrum? therapeutics and tool compounds. Proposed compounds are derived from plant material, endophytic fungi, and rhizosphere bacteria and have previously demonstrated biological activity. Central to the efficient and concise strategies proposed is the knowledge gained in our previously described total syntheses which provides key intermediates for analog development. The final thrust will investigate both the biological target and properties of the tool compounds both in single species and multispecies biofilms. This approach will employ a combination of genetic and MS-proteomic techniques to develop a candidate list of proteins, from which we will identify the targets by biochemical studies. Previous research has demonstrated that these sources have provided compounds with unprecedented biological activity with significant implications to improving human health. Furthermore, they act on therapeutic targets that were previously unknown providing new approaches to combat bacteria.